High resolution 3D reconstruction of fibroblast traction in reconstituted collagen gels. Dense fibrous connective tissues, such as tendon, periosteum and the periodontal ligament, consist of two general components: extracellular matrix and cells. The matrix is composed largely of collagen, whose density, fibril diameter and orientation are the chief determinants of the tissue's mechanical properties. Fibers tend to be aligned parallel to the primary axis of tissue strain, which obviously improves resistance to tensile stresses. The etiology of this organization remains unknown. The cellular component of fibrous connective tissue is mainly fibroblasts which are responsible for both secreting and degrading collagen. Furthermore, fibroblasts have been shown to exert large forces on the matrix through which they crawl, far greater than that of faster moving cells such as leukocytes (Harris et al 1981) This excess force generated through the cytoskeleton brings about a phenomenon known as "traction." Gross examination in two dimensions, reveals that traction by tissue explant s can contract and align local collagen fibers, which may be important for connective tissue morphogenesis and maintenance of tissue integrity (Stopak and Harris, 1982). We have been imaging fibroblasts in collagen using differential interference contrast optics and confocal reflectance contrast microscopy in order to dissect the cellular structures and events involved in 3D fibroblast traction. Solubilized Type I collagen is "re-constituted" into collagen gels composed of a meshwork of collagen fibrils, ranging from 50-IOOnm in diameter, and detectable by reflectance (Friedl, 1997). The gels will be seeded with mouse dermal fibroblasts microinjected with rhodamine-labeled tubulin to a final concentration of 5nM. This low concentration (0.025% of the endogenous tubulin pool) yields "speckled" microtubules (Waterman-Storer and Salmon, 1998) which facilitate visualization of filament dynamics. Cells can also be pre-labeled with non-blocking fluorophore-linked anti-plintegrin antibody (Friedl, 1997). This setup will allow visualization of the three key elements involved in traction: the cytoskeleton, the extracellular matrix and the integrins that bridge the two. The challenges will be in: 1) simultaneously imaging these components, 2) resolving individual collagen fibrils and microtubule speckles in the crowded focal contact region, and 3) continuing visualization of individual living cells over sufficient time to observe tissue reorganization, without photobleaching or photodamaging cells.